PROJECT SUMMARY (PROJECT 3) Genomic and epigenomic instability plays a contributing role in aging. Genomic instability arises from chemical and physical damage to DNA as well as from errors in DNA replication and repair. Recently, activation of endogenous retrotransposable elements has emerged as another major source of age-related genomic instability. SIRT6 plays an important role in maintaining genomic stability by regulating DNA repair. Recent studies by our laboratory demonstrated that SIRT6 has another function in maintaining genome stability by repressing LINE-1 (L1) transposable elements. We showed that SIRT6 represses L1 by mono-ADP ribosylating KAP1, which promotes its interaction with heterochromatin factor HP1. Sirt6 knockout mice display premature aging with a lifespan of 3-4 weeks, associated with dramatic L1 activation. Remarkably, our preliminary data suggests that repression of L1 retrotransposition extends lifespan of these mice. Upon DNA damage SIRT6 vacates L1 promoters and relocalized to DNA damage sites. The dual function of SIRT6 in DNA damage and L1 repression may provide a mechanism for age-related genomic instability. We hypothesize that with aging SIRT6 relocalizes from L1 promoters to the sites of DNA damage and shortened telomeres leading to L1 activation and genome destabilization. The goal of this project is to understand the molecular mechanism of L1 repression by SIRT6 and to dissect the interplay between DNA damage, L1 activation and aging. We will pursue the following specific aims. (1) To determine the mechanism of L1 repression by SIRT6 on the molecular level we will identify the KAP1 amino acids mono-ADP-ribosylated by SIRT6 and determine how this modification affects the binding and recruitment of other heterochromatin factors to L1 promoters. In collaboration with Project 1 we will examine the interaction between SIRT6 and pRb L1 repression pathways. (2) To understand the interplay between SIRT6 function in DNA repair and L1 repression we will perform ChIP-seq to determine SIRT6 localization in young and old mice and in human fibroblasts under basal conditions and after DNA damage. To identify factors that regulate SIRT6 localization on chromatin we will identify posttranslational modifications that direct SIRT6 to either L1 promoters or DNA damage sites. (3) To examine the contribution of L1 activation to the aging process we will treat wild type, Sirt6-late onset, brain-specific, and full body knockout mice with reverse transcriptase inhibitors and test whether the treatment alleviates aging-related phenotypes (with Core C). In collaboration with Core B we will perform TIP-seq or single-cell TIP-seq on the Sirt6 knockout mice to quantify the retrotransposition events before and after treatment. To determine which molecular functions of SIRT6 are involved in longevity we will breed Sirt6 knockout mice with mice harboring SIRT6 separation of function mutations. Finally, we will collaborate with Project 2 to determine the effect of SIRT6 on retrotransposition and lifespan in Drosophila.